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Extracting kinetic parameters for homogeneous [Os(bpy)2ClPyCOOH]+ mediated enzyme reactions from cyclic voltammetry and simulations.

Flexer V, Ielmini MV, Calvo EJ, Bartlett PN - Bioelectrochemistry (2008)

Bottom Line: Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1).The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface.At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.

View Article: PubMed Central - PubMed

Affiliation: INQUIMAE, Departamento de Quimica Inorganica, Analitica y Quimica Fisica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EHA, Argentina.

ABSTRACT
The homogeneous reaction between glucose oxidase and osmium bipyridine-pyridine carboxylic acid in the presence of glucose has been studied in detail by cyclic voltammetry and digital simulation. Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1). The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface. At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.

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Related in: MedlinePlus

Plot of current as a function of glucose concentration in case VI. mΣ = 1.02 mM, eΣ = 1.6 μM in NaH2PO4/Na2HPO4 0.1 M + 0.1 M NaCl buffer solution. Full line shows the best fit to Eq. (6) of the experimental data. Every data point corresponds to a new buffer/mediator/GOx/d-glucose solution that was discarded after taking the experimental CV.
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fig6: Plot of current as a function of glucose concentration in case VI. mΣ = 1.02 mM, eΣ = 1.6 μM in NaH2PO4/Na2HPO4 0.1 M + 0.1 M NaCl buffer solution. Full line shows the best fit to Eq. (6) of the experimental data. Every data point corresponds to a new buffer/mediator/GOx/d-glucose solution that was discarded after taking the experimental CV.

Mentions: To avoid the time dependent effects of the enzyme inactivation processes, we redesigned the experimental protocol so that all the glucose concentration data points were measured after the same time. We freshly prepared new enzyme, Os complex and buffer solution, added glucose to the desired concentration, then measured the voltammogram and discarded the solution. In this way, the enzyme was in contact with the substrate for the shortest time possible in all cases. Fig. 6 shows the new calibration plot obtained in this way. There is greater dispersion of the data points in Fig. 6 than in Fig. 2, due to slight variations in concentration because new solutions were prepared for every data point. However, it is noticeable that the data in Fig. 6 no longer shows evidence for a fall in current at high glucose concentration and is in much better agreement with the proposed model given in Eq. (6) as can be seen from Fig. 6. Notice also that larger current densities than in Fig. 2 are observed for the same glucose concentrations.


Extracting kinetic parameters for homogeneous [Os(bpy)2ClPyCOOH]+ mediated enzyme reactions from cyclic voltammetry and simulations.

Flexer V, Ielmini MV, Calvo EJ, Bartlett PN - Bioelectrochemistry (2008)

Plot of current as a function of glucose concentration in case VI. mΣ = 1.02 mM, eΣ = 1.6 μM in NaH2PO4/Na2HPO4 0.1 M + 0.1 M NaCl buffer solution. Full line shows the best fit to Eq. (6) of the experimental data. Every data point corresponds to a new buffer/mediator/GOx/d-glucose solution that was discarded after taking the experimental CV.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742741&req=5

fig6: Plot of current as a function of glucose concentration in case VI. mΣ = 1.02 mM, eΣ = 1.6 μM in NaH2PO4/Na2HPO4 0.1 M + 0.1 M NaCl buffer solution. Full line shows the best fit to Eq. (6) of the experimental data. Every data point corresponds to a new buffer/mediator/GOx/d-glucose solution that was discarded after taking the experimental CV.
Mentions: To avoid the time dependent effects of the enzyme inactivation processes, we redesigned the experimental protocol so that all the glucose concentration data points were measured after the same time. We freshly prepared new enzyme, Os complex and buffer solution, added glucose to the desired concentration, then measured the voltammogram and discarded the solution. In this way, the enzyme was in contact with the substrate for the shortest time possible in all cases. Fig. 6 shows the new calibration plot obtained in this way. There is greater dispersion of the data points in Fig. 6 than in Fig. 2, due to slight variations in concentration because new solutions were prepared for every data point. However, it is noticeable that the data in Fig. 6 no longer shows evidence for a fall in current at high glucose concentration and is in much better agreement with the proposed model given in Eq. (6) as can be seen from Fig. 6. Notice also that larger current densities than in Fig. 2 are observed for the same glucose concentrations.

Bottom Line: Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1).The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface.At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.

View Article: PubMed Central - PubMed

Affiliation: INQUIMAE, Departamento de Quimica Inorganica, Analitica y Quimica Fisica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EHA, Argentina.

ABSTRACT
The homogeneous reaction between glucose oxidase and osmium bipyridine-pyridine carboxylic acid in the presence of glucose has been studied in detail by cyclic voltammetry and digital simulation. Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1). The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface. At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.

Show MeSH
Related in: MedlinePlus